The roles of NLRP3 and prostaglandin E2 in mediating bidirectional cross-talk between bovine endometrial fibroblasts and M2 macrophages.

The bovine endometrium undergoes dynamic structural and functional changes during the estrous cycle, driven by intricate cellular interactions and a complex network of chemokines, cytokines, and growth factors. Among the key cell types, endometrial stromal fibroblasts and M2 macrophages are essential for anti-inflammatory responses and tissue remodeling. However, their bidirectional cross-talk remains poorly characterized. In this study, we investigated fibroblast-macrophage interactions using a conditioned medium-based co-culture system. M2 macrophages were treated with myofibroblast-conditioned medium (MFbCM), while myofibroblasts were treated with M2 macrophage-conditioned medium (M2ø CM), allowing detailed analysis of reciprocal regulatory effects. M2ø CM significantly upregulated fibroblast expression of fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor (VEGF), and matrix metalloproteinase-9 (MMP-9), suggesting M2 macrophage-mediated modulation of fibroblast remodeling activity. Conversely, MFbCM enhanced interleukin-10 (IL-10) and arginase expression in M2 macrophages, supporting fibroblast-driven polarization. Notably, both conditioned media increased expression of NOD-like receptor family pyrin domain containing 3 (NLRP3) in target cells. Inhibition of NLRP3 using the selective inhibitor MCC950 revealed that the NLRP3-prostaglandin E2 (PGE2) axis plays a pivotal role in mediating the cross-talk between these cell types. Collectively, our findings reveal a pivotal NLRP3-PGE2 signaling axis in the regulation of fibroblast-macrophage interactions, offering novel insights into the mechanisms underlying bovine endometrial homeostasis and remodeling.